Isoflavonoids represent a class of secondary metabolites produced in legumes by a branch of the phenylpropanoid pathway and include such compounds as isoflavones, isoflavanones, rotenoids, pterocarpans, isoflavans, quinone derivatives, 3-aryl-4-hydroxy-coumarins, 3-arylcoumarins, isoflav-3-enes, coumestans, alpha-methyldeoxybenzoins, 2-arylbenzofurans, isoflavanol, coumaronochromone and the like. In plants, these compounds are known to be involved in interactions with other organisms and to participate in the defense responses of legumes against phytopathogenic microorganisms (Dewick, P. M. (1993) in The Flavonoids, Advances in Research Since 1986, Harborne, J. B. Ed., pp. 117–238, Chapman and Hall, London). Isoflavonoid-derived compounds also are involved in symbiotic relationships between roots and rhizobial bacteria which eventually result in nodulation and nitrogen-fixation (Phillips, D. A. (1992) in Recent Advances in Phytochemistry. Vol. 26, pp 201–231, Stafford, H. A. and Ibrahim, R. K., Eds, Pleneum Press, New York), and overall they have been shown to act as antibiotics, repellents, attractants, and signal compounds (Barz, W. and Welle, R. (1992) Phenolic Metabolism in Plants, pg 139–164, Ed by H. A. Stafford and R. K. Ibrahim, Plenum Press, New York).
Isoflavonoids have also been reported to have physiological activity in animal and human studies. For example, it has been reported that the isoflavones found in soybean seeds possess antihemolytic (Naim, M., et al. (1976) J. Agric. Food Chem. 24:1174–1177), antifungal (Naim, M., et al. (1974) J. Agr. Food Chem. 22:806–810), estrogenic (Price, K. R. and Fenwick, G. R. (1985) Food Addit. Contam. 2:73–106), tumor-suppressing (Messina, M. and Barnes, S. (1991) J. Natl. Cancer Inst. 83:541–546; Peterson, G., et al. (1991) Biochem. Biophys. Res. Commun. 179:661–667), hypolipidemic (Mathur, K., et al. (1964) J. Nutr. 84:201–204), and serum cholesterol-lowering (Sharma, R. D. (1979) Lipids 14:535–540) effects. These epidemiological studies indicate that isoflavones in soybean protein products, when taken as a dietary supplement, may produce many significant health benefits.
Free isoflavones rarely accumulate to high levels in soybeans. Instead they are usually conjugated to carbohydrates or organic acids. Soybean seeds contain three types of isoflavones in four different forms: the aglycones, daidzein, genistein and glycitein; the glucosides, daidzin, genistin and glycitin; the acetylgucosides, 6″-O-acetyldaidzin, 6″-O-acetylgenistin and 6″-O-acetylglycitin; and the malonylglucosides, 6″-O-malonyldaidzin, 6″-O-malonylgenistin and 6″-O-malonylglycitin. In accordance with the present invention, all of these compounds are included in the term isoflavonoids. The content of isoflavonoids in soybean seeds is quite variable and is affected by both genetics and environmental conditions such as growing location and temperature during seed fill (Tsukamoto, C., et al. (1995) J. Agric. Food Chem. 43:1184–1192; Wang, H. and Murphy, P. A. (1994) J. Agric. Food Chem. 42:1674–1677). In addition, isoflavonoid content in legumes can be stress-induced by pathogenic attack, wounding, high UV light exposure and pollution (Dixon, R. A. and Paiva, N. L. (1995) Plant Cell 7:1085–1097).
The biosynthetic pathway for isoflavonoids in soybean and their relationship with several other classes of phenylpropanoids is presented in FIG. 1. Many of the enzymes involved in the synthesis of isoflavonoids in legumes have been identified and many of the genes in the pathway have been cloned. These include three P450-dependent monooxygenases, cinnamate 4-hydoxylase (Potts, J. R. M., et al. (1974) J. Biol. Chem. 249:5019–5026), isoflavone 2′-hydroxylase (Akashi, T. et al. (1998) Biochem. Biophys. Res. Commun. 251:67–70), and dihydroxypterocarpan 6a-hydroxylase (Schopfer, C. R., et. al. (1998) FEBS Lett. 432:182–186). However, to date the gene encoding isoflavone synthase, the first step in the phenylpropanoid branch that commits metabolic intermediates to the synthesis of isoflavonoids, has been neither identified nor cloned from any species. In this central reaction, 2S-flavanone is converted into an isoflavonoid such as genistein and daidzein. The enzymatic reaction for this oxidative aryl migration step was first reported by Hagmann, M. L. and Grisebach, H. ((1984) FEBS Lett. 175.199–202). The reaction involves a P450 monoxygenase-mediated conversion of the 2S-flavanone to a 2-hydroxyisoflavanone, followed by conversion to the isoflavonoid. This last step is possibly mediated by a soluble dehydratase (Kochs, G. and Grisenbach, H. (1985) Eur. J. Biochem. 155:311–318). However, the 2-hydroxyisoflavanone intermediate was described as unstable and could convert directly to genistein.
Cytochrome P450-dependant monooxygenases comprise a large group of heme-containing enzymes, most of which catalyze NADPH- and O2-dependant hydroxylation reactions. Most of these enzymes do not use NADPH directly, but rely upon an interaction with a flavoprotein known as a P450 reductase that transfers electrons from the cofactor to the P450. Cloning of plant P450s by traditional protein purification strategies has been difficult, as these membrane-bound proteins are often very unstable and are typically present in low abundance. PCR-based cloning strategies using sequence homologies between P450s has increased dramatically the number of P450 genes cloned. However, the in vivo activity of many of these cloned genes remains unknown and they are classified simply as P450s, and are grouped into families based solely on sequence homology (Chapple, C. (1998) Annu. Rev. Plant Physiol. Plant Mol. Bio. 49:311–343). Proteins that are greater than 55% identical are designated as members of the same subfamily, while P450s that are 97% identical, or greater, are assumed to be allelic variants of the same gene (Chapple, C. (1998) Annu. Rev. Plant Physiol. Plant Mol. Bio. 49:311–343).
Efforts to determine in vivo activities of existing P450 clones are increasing. Most efforts involve expressing genes or cDNAs for P450s in yeast or insect cell systems, and then screening for a particular activity. For example, isoflavone 2′-hydroxylase (Akashi, T., et al. (1998) Biochem. Biophys. Res. Commun. 251:67–70) and dihydroxypterocarpan 6a-hydroxylase (Schopfer, C. R., et al. (1998) FEBS Letters 432:182–186) were identified in this manner.
The physiological activities associated with isoflavonoids in both plants and humans makes the manipulation of their contents in crop plants highly desirable. For example, increasing levels of isoflavonoid in soybean seeds would increase the efficiency of extraction and lower the cost of isoflavone-related products sold today for use in either reduction of serum cholesterol or in estrogen replacement therapy. Decreasing levels of isoflavonoid in soybean seeds would be beneficial for production of soy-based infant formulas where the estrogenic effects of isoflavonoid are undesirable. Raising levels of isoflavonoid phytoalexins in vegetative plant tissue could increase plant defenses to pathogen attack, thereby improving plant disease resistance and lowering pesticide use rates. Manipulation of isoflavonoid levels in roots could lead to improved nodulation and increased efficiencies of nitrogen fixation. To date, however, it has proven difficult to develop soybean or other plant lines with consistently high levels of isoflavonoid. Because isoflavone synthase is the central reaction in pathways producing isoflavonoids, identification of this functional gene is extremely important, and its manipulation via molecular techniques is expected to allow production of soybeans and other plants with high, stable levels of isoflavonoid. Introduction of the isoflavone synthase gene in non-legume crop species including, but not limited to, corn, wheat, rice, sunflower, and canola could lead to synthesis of isoflavonoids. The expression of isoflavonoids would confer to these species disease resistance and/or properties which produce human/livestock health benefits.
Substrates for isoflavone synthase may be limiting for synthesizing very high levels of isoflavonoids in soybean, or for synthesizing isoflavonoids in non-legumes. It is desirable to increase the flux of metabolites through the phenylpropanoid pathway to provide additional amounts of substrate to those occurring naturally. Different stress conditions such as UV irradiation, phosphate starvation, prolonged exposure to cold, and chemical (such as herbicide) treatment can cause activation of the phenylpropanoid pathway. While these treatments may produce the desired substrate availability, it is more desirable to have a genetic means of activating the phenylpropanoid pathway. It is known that expression of genes encoding certain transcription factors can regulate the expression of various genes that encode enzymes of the phenylpropanoid pathway. These include, but are not limited to, the C1 myb-type transcription factor of maize and the AmMyb305 of Antirrhinum majus. The C1 myb-type transcription factor of maize, in conjunction with the myc-type transcription factor R, activates chalcone synthase and chalcone isomerase genes (Grotewold, E., et al. (1998) Plant Cell 10:721–740). The Antirrhinum majus AmMyb305 activates the phenylalanine ammonia lyase promoter (Sablowski, R. W., et al. (1994) EMBO J. 13:128–137). Transcription factors such as these may be expressed in host plant cells to activate expression of genes in the phenylpropanoid pathway thereby increasing the encoded enzyme activities and the flux of compounds through the pathway. Increases in the precursors to substrates of isoflavone synthase would enhance the production of isoflavonoids.